A comprehensive and longitudinal evaluation of the different populations of lymphoid and myeloid cells in the peripheral blood of patients treated with chemoradiotherapy for head and neck cancer

Background Immunotherapy provided significant survival benefits for recurrent and metastatic patients with head and neck cancer. These improvements could not be reproduced in patients treated with curative-intent chemoradiotherapy (CRT) and the optimal radio-immunotherapy (RIT) concepts have yet to be designed. Exploration and analysis of the pre-therapeutic immune status of these patients and the changes occurring during the treatment course could be crucial in rationally designing future combined treatments. Methods Blood samples were collected from a cohort of 25 head and neck cancer patients treated with curative-intended (C)-RT prior to therapy, after the first week of treatment, and three months after treatment completion. Peripheral blood mononuclear cells (PBMCs) or all nucleated blood cells were isolated and analyzed via flow cytometry. Results At baseline, patients showed reduced monocyte and lymphocyte counts compared to healthy individuals. Although overall CD8+ T-cell frequencies were reduced, the proportion of memory subsets were increased in patients. Radiotherapy (RT) treatment led to a further increase in CD8+ effector memory T-cells. Among myeloid populations, tumor-promoting subsets became less abundant after RT, in favor of pro-inflammatory cells. Conclusion The present study prospectively demonstrated a complex interplay and distinct longitudinal changes in the composition of lymphocytic and myeloid populations during curative (C)-RT of head and neck cancer. Further validation of this method in a larger cohort could allow for better treatment guidance and tailored incorporation of immunotherapies (IT) in the future. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-024-03810-6.


Introduction
Squamous cell carcinoma of the head and neck (SCCHN) accounts for 6% of the total incidence of malignant tumors, and about 500,000 new patients are diagnosed each year around the world [1].Definitive chemoradiotherapy (CRT) or radical surgery, i.e., resection of the primary tumor and dissection of regional lymph nodes, followed by adjuvant radiotherapy (RT) or CRT, are routinely performed in locoregionally advanced SCCHN leading to a 5-year overall survival rates of 40-60% [2,3].Many advances regarding tumor control and patient survival have been made over the past decades [4].
The advent of immunotherapy (IT) provided significant survival benefits for recurrent and metastatic SCCHN patients for the first time since years [5].Yet, these improvements could not be reproduced in patients treated with curative-intent CRT [6], and the optimal radio-immunotherapy (RIT) concepts are yet to be designed [7].Thus, thorough exploration and analysis of the pre-therapeutic immune status of these patients and the changes occurring during the treatment course could be crucial in rationally designing combined treatments in the future.
As an exploratory, ancillary study to a single-center prospective trial evaluating the benefits of adaptive RT for stage II-IVB SCCHN (NCT03972072), venous blood from patients treated within the trial was collected and analyzed using flow cytometry.In the context of CRT for SCCHN, both the innate and adaptive immune system and local tumor infiltrating and peripheral blood immune cells play a key role for patients' outcome [8,9].However, most studies regarding blood-based biomarkers have exclusively assessed only pre-treatment liquid biopsies for their prognostic impact, and only included non-immune biomarkers [9].A comprehensive profiling of all major immune cell subpopulations was not performed before.
The aim of this pilot study was to longitudinally evaluate different populations of lymphoid and myeloid cells in the peripheral blood of patients treated with definitive (C)-RT for head and neck cancer in order to assess abundances and relative changes of these cell types occurring during the treatment course from baseline to follow-up.

Patient population and treatment
The MARTHA trial is an ongoing, single-center, prospective, open label, single-arm study investigating a possible reduction in late-term toxicity for patients with newly diagnosed and histologically confirmed SCCHN of the oral cavity, oro-, hypopharynx or larynx in the UICC stages II-IVB treated with MR-guided, adaptive standarddose bilateral (C)-RT.Further, inclusion requires an ECOG Performance score < 2 (Eastern Cooperative Oncology Group) and complete staging including FDG PET-CTscan (fluorodeoxyglucose positron emission tomographycomputed tomography), contrast enhanced magnetic resonance imaging (MRI), or computed tomography (CT) prior to registration.Normo-fractionated RT consists of 2 Gray (Gy) single-dose and 70 Gy cumulative dose to macroscopic tumor (planned target volume (PTV)-70 Gy: primary and involved nodes), 60 Gy to involved nodal levels (PTV-60 Gy) and 50 Gy for elective nodal irradiation (PTV-50 Gy).Concurrently, standard platinum-based chemotherapy (CT) is administered with Cetuximab as an alternative for platinum-ineligible patients.
Treatment is applied with an MRI-Linac (MRIdian®, ViewRay, OH, USA) with a pre-defined image-guided RT and weekly plan adaptation protocol in order to achieve superior normal tissue sparing.The primary endpoint of the study is the rate of radiation-induced xerostomia of grade 2 or worse 12 months after treatment (NCT03972072).The prospective longitudinal evaluation of the peripheral immune cell subpopulations is an ancillary study performed for the first 25 patients included in the trial.Written informed consent for the trial and the present translational project was given by all study participants.The study was approved by the ethics committee of the University Hospital of Zurich, Switzerland (BASEC No. 2019-00993).The results of the peripheral blood analyses at baseline were further compared to those collected from healthy donors.

Translational analysis
For the translational study, blood from a healthy cohort (HC) was collected within the "Fundamental research project for phenotypical and functional characterization of different leukocyte subsets in healthy and diseased individuals" (PFCL-1, BASEC No. 2016-01440) which has been reviewed and approved by the competent Swiss authorities and has been carried out in accordance with the current version of the Declaration of Helsinki, the guidelines of Good Clinical Practice, and Swiss legal requirements.The HC was defined as individuals without known active acute or chronic diseases at the time of blood donation.
This study was performed by withdrawing 20 ml of blood in heparin vacutainer tubes (BD Healthcare) from the tumor patient cohort (TC) at pre-defined time points: Prior to therapy (1), after the first week of treatment (2), and three months after treatment completion (3).The blood was processed within one hour of collection, stained and analyzed by flow cytometry in the same day for all three time points.
Peripheral blood mononuclear cells (PBMCs) or nucleated blood cells were isolated using the traditional Ficoll gradient centrifugation or HetaSep according to the manufacturer's protocol, respectively.Briefly, for Ficoll isolation 10 ml blood was diluted 1:1 with DPBS (Gibco) and layered on 15 ml of Ficoll Paque Plus (GE Healthcare) at a 45° angle.The tubes were centrifugated at 400 g for 20 min Acc:4, Dec:0 at 20 °C.The PBMC layer was collected and washed with DPBS before staining for flow cytometry analysis.For HetaSep isolation, 10 ml blood was mixed with 2 ml HetaSep (Stemcell) and centrifuged at 100 g for 10 min Acc:9, Dec:2 at 20 °C.Upper layer containing nucleated blood cells were collected and washed with DPBS before staining for flow cytometry analysis.
The isolated cells were stained in FACS (fluorescence activated cell sorting) buffer containing the appropria te fluorescently labeled monoclonal antibodies (mAbs) on ice for 20 min (Supplementary Table 6).Cell viability was assessed by staining with Fixable Viability Dye eFluor™ 780 (Invitrogen).Intracellular staining was performed using Foxp3/transcription factor staining buffer set (Invitrogen) following manufacturers' instructions.Briefly, samples were fix/permed for 1 h at 4 °C and stained with intracellular staining prepared in 1X wash buffer for 30 min at room temperature.Samples were acquired using BD LSR Fortessa on the same day.

Statistical analysis
Routine blood samples from patients were compared using a paired Wilcoxon test with SPSS (IBM SPSS Statistics, V29.0, Armonk, NY, USA).Flow data was analyzed by manual gating using FlowJo software and R Studio (R Studio: Integrated Development for R. RStudio, PBC, Boston, MA).All samples were gated following the same hierarchy (Supplementary Figs.1-3), while the individual gates were adjusted for different samples.The population gating was mainly done visually.For certain markers where the distinction between the positive and negative is less clear, such as CD206, CCR5 and CXCR3, fluorescence minus one (FMO) controls were used.Statistics comparing the HC with the patients were done using an unpaired Wilcoxon test, whereas the statistics comparing the patient visits among each other were done using a paired Wilcoxon test.Paired and unpaired tests were normalized and corrected for multiple testing, separately.For all statistical analyses, significance was accepted at the 95% confidence level (p < 0.05).For data visualization, Adobe Illustrator (Adobe Inc.Illustrator, V27.3.1,DE, USA) was utilized.

Patients' characteristics, treatment and outcome
Twenty-five patients of the MARTHA trial receiving definitive (C)-RT were included into this study.The majority of the patients were men (80%), and the median age was 61 years.All patients were in good general condition of an ECOG performance status 0 (84%) or 1 (16%).Most patients had a history of smoking (80%) and/or alcohol (72%) abuse.Primary tumors were mostly located in the oropharynx (68%).In total, 44% of the tumors were p16-positive and 16% had bilateral neck node-involvement.All patients underwent primary RT, mostly with concurrent platinumbased CT (Table 1).
After a median follow-up of 25 months (range 11-27), seven (28%) patients were diagnosed with local or distant recurrences.Two (8%) patients had died following tumor progression.

Analysis of myeloid cell populations
There was a significant imbalance between the relative amount of myeloid (p = 0.004) cell populations when healthy individuals were compared with the SCCHN patients, with tumor patients showing generally increased levels of myeloid cells (Supplementary Fig. 1).
Within the myeloid compartment, eosinophil granulocytes were generally less abundant in the TC compared to the HC (p < 0.001), while the proportions of neutrophil granulocytes and basophil granulocytes were not significantly altered.The same applied for monocytes.The proportion of classical and plasmacytoid dendritic cells (cDC, pDC) were not changed in comparison with HC and during therapy but their proportion within the myeloid cells were increased during follow-up (p = 0.010 and p = 0.026, respectively) (Fig. 1).
Within the monocytes, the proportion of classical monocytes was decreased in the TC compared to the HC (p < 0.001).In contrast, non-classical monocytes were more abundant in the TC (p < 0.001).Classical monocytes can differentiate into macrophages in tissue and can acquire anti-or pro-tumorigenic phenotypes.CCR5 and CD206 expressions were assessed on monocytes as an indicator of maturation and differentiation into an M2 phenotype, respectively.CD206 expression was more abundant within CCR5 + monocytes in the TC compared to the HC, where its expression was virtually absent (Fig. 2, Supplementary Fig. 2).
Regarding the ratio of neutrophil granulocytes to CD3 + T lymphocytes, there was no difference when the TC was compared to the healthy individuals or between the three measurements for the TC (Supplementary Fig. 3).

Analysis of lymphoid cell populations
Within the PBMCs, the percentage of total T-cells was declined during therapy and follow-up (p = 0.022) which was caused by a reduction in conventional CD4 + T-cells (p = 0.002).CD8 + T-cell percentages were shown to be almost twofold decreased in the TC (p = 0.020), which was not altered by therapy.Regarding NK cells, the frequency of CD56 bright NK cells was decreased prior to therapy in TC compared to the HC (p = 0.002), whereas the frequencies of CD56 dim NK cells did not differ.CD56 bright NK cells were decreased within the PBMCs also during therapy followed   3, Supplementary Fig. 4).CD8 + T lymphocytes can be further subclassified according to their expression of CCR7 and CD45RA.While naive T-cells (Tn) were generally less abundant in the TC compared to the HC (HC vs. TP1: p = 0.003; HC vs. TP1-3: p < 0.001), effector cells such as T-effector memory (Tem), T central memory (Tcm) and TEMRA were more abundant in the TC.This remained unchanged during therapy as well as during follow-up (Fig. 4a-d, Supplementary Fig. 5).
Furthermore, for CD8 + T-cells, CXCR3 is a key chemokine receptor required for migration into the tissue.The expression of this marker was reduced for the subset of Tcm cells (HC vs. TP1: p = 0.025; HC vs. TP1-3: p = 0.039) but unchanged for Tem, Tn, and TEMRA cells in the TC compared to the HC (Fig. 4e-h).

Quantitative peripheral blood counts from routine tests
Prior to therapy, during RT and at follow-up visits, patients routinely received peripheral blood withdrawals.C-reactive protein (CRP)/albumin ratio was significantly reduced following therapy when compared to the first week of treatment (p = 0.023), whereas the ratio of neutrophil/ lymphocyte (N/L) significantly increased during and after therapy when compared to the baseline levels (p = 0.010 and 0.013).Furthermore, hemoglobin, platelets, and leukocytes were decreased three months after therapy compared to the baseline levels (p values: 0.001, < 0.001, and < 0.001, respectively; Supplementary Table 2).

Discussion
Although there is emerging evidence about the role of both myeloid and lymphoid immune cells in promotion, response to therapy and recurrence of head and neck cancer, most data originate from tumor infiltrating/ -associated populations in the tissue.Little is known both about the possible impact of immune cells in the peripheral blood and their longitudinal changes during CRT [10].Even less data exist regarding specific subpopulations, other than the commonly studied neutrophil granulocytes and CD4 + /CD8 + lymphocytes.The aim of this exploratory study was not only to provide a detailed descriptive analysis of all the important subpopulations of the innate and adaptive immune system found in the peripheral blood of patients with SCCHN, but also to compare them to those of healthy individuals and identify significant changes The present analysis demonstrated a series of interesting findings:

Comparison of baseline values to healthy individuals
There was a significant imbalance between the relative amount of lymphocytic and myeloid populations when healthy individuals were compared with the SCCHN patients: Tumor patients showed generally increased levels of myeloid cells and decreased T-lymphoid cells.This can be interpreted as a surrogate for the impaired or altered immunity as a reason or result of tumorigenesis.Interestingly, even the absolute lymphocyte count alone has been shown to be predictive for outcome in patients receiving combined IT and RT before [11].This has to be taken into account, especially in the case of irradiation of large volumes of peripheral blood, as it is the case with current recommendations for elective RT volumes of the neck, encompassing both carotid arteries [12].Such "traditional" approaches might hamper the overall treatment effect and have to be critically reconsidered in the future [13].Importantly, in the present study, all of the patients received a bilateral RT of the neck lymphatics.Nevertheless, the impact of lymphopenia in relationship to the effects of a curative RT treatment without concomitant IT remains controversial.As was shown by some authors before, it is still unknown if RT-induced peripheral/systemic lymphopenia is of any relevance for the patients' outcome compared to the decreasing cells after RT of regional lymph nodes [14].It is possible that peripheral lymphopenia is an epiphenomenon and less relevant for treatment outcome.

Myeloid cells
First, regarding myeloid cells populations, the significant reduction in classical monocytes, in the TC and after the follow-up time point could be an important finding, as these cells or their direct derivatives in the tissue are generally considered immunosuppressive and their numbers inversely correlate with those of CD8 + cells [15,16].The opposite was the case with non-classical monocytes, whose numbers significantly increased after treatment.Little is known about this cell-type, but some authors suggest a positive role in recruiting CD8 + cells and thus promoting tumor cell killing [17].Intriguingly, no significant findings were observed for the neutrophil granulocytes frequencies, neither when comparing the HC to the SCCHN patients, or after longitudinal assessment, despite the fact, that there was a slight but significant increase in the N/L ratio over time.
Neutrophile to Lymphocyte ratioThis N/L ratio has been described as a prognostic factor by several authors and even meta-analyses demonstrated an increased hazard ratio for impaired outcome for a ratio of 2-5 [18,19].In a previous single-center, but large cohort of patients with laryngeal carcinoma receiving either definitive or adjuvant RT, a higher baseline N/L ratio significantly correlated with an impaired outcome [20].Our cohort was too small and only few recurrence events were observed for a correlation with oncological endpoints.The median value observed in the SCCHN patients included in this study for all time points exceeded a value of 3. Nevertheless, some significant increase in both the ratio and the relative neutrophil granulocyte numbers observed compared to baseline, during treatment and at the first follow-up could be an artifact, attributed to the higher resistance to CT of neutrophil granulocytes and the delayed recovery of lymphocytes after CT compared to neutrophil granulocytes populations.

Lymphatic cells
CD8 + effector memory cell rates significantly increased during treatment and at follow-up compared to baseline for treated patients, revealing the immunogenic role of RT in promoting an adaptive response [21].Recently published data elucidate that the exposure of neo-antigens induced by RT lead to priming and recruitment of CD8 + T-cells [22,23].Based on that, RT can be used synergistically with immunotherapeutic approaches such as adoptive T-cell treatments in solid tumors [24].Interestingly, the same temporal change was observed for NK cells.CD56 bright NK cells, as separately examined here, have different functions than regular NK cells, as they are less cytotoxic and more cytokine producing.However, this specific differentiation has not often been examined in correlation with CRT.NK cell dysfunction is known to promote tumorigenesis and treatment resistance [25][26][27].Moreover, NK cells play a key role in RT-induced inflammation and treatment efficacy [28], and there already exist methods to enhance their effects, e.g., via ataxia telangiectasia and Rad3-related inhibitors (ATRi) [29].On the other hand, the previously observed phenomenon of RT-induced upregulation of T-regulatory cells (Tregs) in the tissue was not significant here in the blood of irradiated patients, indicating that this possible mechanism of acquired resistance to standard treatment is mostly tissue based.Taken together, the kinetics of different lymphocyte populations in peripheral blood and especially those of the NK cells can serve might be an easy-to-assess predictive tool for response after curative-intended (C)-RT.Niu et al. [30] could already demonstrate this in a small cohort of patients with head and neck cancer with a longer follow-up compared to the present cohort.Regarding the observed differences between the HC and TC, already in the first stages of tumorigenesis, the T-cell response can eliminate cancer cells expressing recognized antigens.Nevertheless, some cancer cells may develop alterations to evade the immune system by immunosuppressive mechanisms that hamper the capability of T-cells to recognize and eliminate them [31].Such immune-resistant cells acquire selection benefits and lead to tumor progression.Moreover, and although no patients with known immunodeficiency syndromes were recruited for this study, a preexisting impairment of the adaptive immune response can also be a reason for cancer development and progression.Patients with deficient T-cell responses or reduced T-cell levels might be in need of non-Tcell-addressing therapies, such as M2 macrophage depletion or NK cell therapies [32].Both a lower than normal lymphocyte count at baseline and a reduced NK cell activity have been described before for these patients [33,34].Upregulation of tumor-promoting/immune suppressive Tregs in the tissue following RT is a well-described phenomenon that could negatively influence the efficacy of treatments [35,36], also specifically in SCCHN patients [37].Importantly, a significant and continuous increase in Tregs in the peripheral blood of SCHNN was observed here.Novel approaches investigate the combination of RT and Tregs-depletion as a possible promising treatment method, which can be also combined with immune-checkpoint-inhibition (ICI) to further enhance the effects [38].In the recurrent/metastatic setting, in patients treated with IT, Gavrielatou et al. demonstrated that assessment of different pre-treatment PBMCs via FACS was able to identify immune cell phenotypes associated with response to ICI [10].More precisely, CD8 + and CD4 + cells with stem-like or exhaustion characteristics as well as CD21 −/low B-cells were predictive for an improved response.However, this phenomenon might be specific to treatment via ICI [39].

Limitations
There are limitations of this study.First of all the relatively low number of patients included and the lack of external validation do not allow for more valid statistical analyses.Biomarker establishment for future trials needs robust correlation with oncological outcomes.However, this was impossible in the current study with only seven events and no specific outliers among these seven patients within the significant cell populations.Moreover, there was no possibility either to perform a multivariate analysis with this limited number of events or to define a statistically meaningful cut-off for the parameters, which would then be arbitrary and overfitted.It should be stressed that the purpose of the study was descriptive, as there is a lack of data describing in such detail and longitudinally mode the peripheral immune cells of a homogeneous, curatively treated cohort of HNSCC patients.Secondly, the CT applied could interfere with the results and the longitudinal changes observed.Third, the mere numbers of immune cells counted and their changes do not always represent the same changes in functionality of these cells.Recent studies revealed polymorphisms, including large heterogeneity in the receptors, signaling and function of several subpopulations of the same cell-type, which can only be assessed by more elaborate methods and can further guide treatment individualization [40].
Nevertheless, this is the only study investigating all major immune cell populations in the peripheral blood, in a prospective and longitudinal manner, implementing state-ofthe art flow cytometry in a homogeneous cohort of patients treated with curative (C)-RT and compared the findings also with a HC.Previous efforts in this field only included recurrent/metastatic patients without RT or evaluated only some major lymphocytic and/or neutrophil granulocyte populations.

Conclusion
The present study prospectively demonstrated the complex interplay and distinct longitudinal changes in the composition of all various lymphocytic and myeloid subpopulations during curative (C)-RT of SCCHN.Major findings were the significantly reduced lymphocyte count already at baseline for untreated patients compared to healthy individuals, the impact of RT treatment to enhance the expression of CD8 + effector memory cells, and finally the differential response of myeloid populations, with canonical, tumor-promoting myelocytes becoming less abundant following, in favor of non-canonical CD8 + recruiting cells.This quick, noninvasive and increasingly simplified approach could allow for a comprehensive evaluation of the patients' current immune status at every step of a continuous treatment, a better treatment guidance and finally tailored incorporation of immune therapies in the future [41].

Fig. 1
Fig. 1 Frequencies of Myeloid Cell Subsets.Abundances of indicated cell subsets were analyzed by flow cytometry and are shown as percentages of myeloid cells.The data of the healthy cohort are compared to three blood collection time points of the tumor patients: TP1: Prior to therapy; TP2: after the first week of treatment; TP3: three months after treatment completion.a-c Abundances of neutro-

Fig. 2
Fig. 2 Frequencies of monocyte subsets.Abundances of indicated cell subsets were analyzed by flow cytometry and are shown as percentages of different monocyte fractions.The data of the healthy cohort are compared to three blood collection time points of the tumor patients: TP1: Prior to therapy; TP2: after the first week of treatment; TP3: three months after treatment completion.a-c fre-

Fig. 3
Fig. 3 Frequencies of lymphoid cells.Abundances of indicated cell subsets were analyzed by flow cytometry and are shown as percentages of peripheral blood mononuclear cells (PBMCs).The data of the healthy cohort are compared to three blood collection time points of the tumor patients: TP1: Prior to therapy; TP2: after the first week of treatment; TP3: three months after treatment completion.A: Frequencies of B-cells as percentages of PBMCs.B: Frequencies of total T-cells as percentages of PBMCs.C: Frequencies

Fig. 4
Fig. 4 Frequencies of CD8 + T-cell subsets.Abundances of indicated cell subsets were analyzed by flow cytometry and are shown as percentages of peripheral blood mononuclear cells (PBMCs).The data of the healthy cohort are compared to three blood collection time points of the tumor patients: TP1: Prior to therapy; TP2: after the first week of treatment; TP3: three months after treatment completion.a-d Frequencies of central memory (Tcm, CD45RA − CCR7 + ),effector memory (Tem, CD45RA − CCR7 − ), T-effector memory re-

Table 1
Baseline characteristics